Role of copper in photochemical damage to hair.

OBJECTIVE: The objective of this work was to identify whether low levels of redox metals such as copper will accelerate damage to hair on exposure to UV irradiation and whether this damage can be prevented. METHODS: The methods used were proteomics to measure the protein damage via protein loss after different periods of exposure and mass spectroscopy methods to identify specific marker peptides that are specifically created by this type of damage. RESULTS: In this work, we have developed new insights into the mechanism of UV damage using these proteomic methods. A marker fragment in the hair protein loss extract was identified (m/z = 1279) that is unique to UV exposure and increases with time of UV exposure. We have also identified for the first time in hair the role of exogenous copper in increasing UV damage both in terms of total protein degradation and also increased formation of the marker fragment and proposed a mechanism of action. It has been demonstrated that shampoo treatment containing a chelant such as N,N'-ethylenediamine disuccinic acid (EDDS) reduced copper accumulation in hair. CONCLUSION: This work provides evidence for the role of copper in UV-induced damage to hair and strategies to reduce copper levels in hair using a chelant such as EDDS.

Isolated gastrointestinal metastasis of breast carcinoma: a case report.

Purpose. Gastrointestinal tract is one of the rare locations for breast cancer metastasis. This paper shows such metastasis may occur even in the absence of breast metastasis in other more common locations. Case Report. A 64-year old female was admitted to the hospital with abdominal discomfort and diarrhea. She had breast carcinoma treated 7 years previously with normal follow-up since. Colonoscopy showed hepatic flexure thickening that was confirmed to be breast metastasis. Staging investigations showed upper and lower gastrointestinal tract metastasis with negative findings elsewhere. Conclusion. Although more common causes for gastrointestinal symptoms should be excluded, however, a high index of suspicion of metastatic breast cancer is needed when such patients develop gastrointestinal symptoms.

Efficacy and safety of intravenous anti-D immunoglobulin (Rhophylac) in chronic immune thrombocytopenic purpura.

OBJECTIVES: This Phase III study examined the efficacy and safety of Rhophylac (CSL Behring AG, Bern, Switzerland), a highly pure, liquid-stable anti-D preparation, in chronic immune thrombocytopenic purpura (ITP). MATERIALS AND METHODS: Ninety-eight patients (96 adults, two adolescents) with chronic ITP and platelet counts < 30 x 10(9)/l received a single intravenous injection of 50 microg/kg bodyweight Rhophylac. RESULTS: A response (defined as an increase in platelet count by >or= 20 x 10(9)/l to >or= 30 x 10(9)/l in the first 15 days after treatment) was seen in 66% of patients. Mean time to response was 3.1 +/- 3.0 days, and mean duration of response was 19.2 +/- 1.1 days for responders. The most frequent drug-related adverse events were chills, pyrexia, an increase in bilirubin, and headache; events were mainly mild or moderate. there was no severe hemolysis or renal failure. CONCLUSION: rhophylac is well tolerated and efficacious in chronic itp.

Long-term observation of patients with anti-neutrophil cytoplasmic antibody-associated vasculitis treated with rituximab.

OBJECTIVE: Rituximab, a chimeric anti-CD20 monoclonal antibody, has been shown to be quite effective in the treatment of immune disorders resulting from autoantibodies. We prospectively studied the long-term effects of rituximab in 10 patients with anti-neutrophil cytoplasmic antibody (ANCA)-positive vasculitis refractory to conventional therapy (n=3) or in second or subsequent relapse (n=7). METHODS: The median age of patients was 53 yrs (range 38-70 yrs). Eight were classified as Wegener's granulomatosis, and two as microscopic polyangiitis. Clinical activity was assessed using the Birmingham Vasculitis Activity Score modification for Wegener's granulomatosis. Treatment consisted of intravenous infusions of rituximab given at the dose of 375 mg/m2 weekly for four consecutive weeks. RESULTS: All patients experienced a rapid clinical improvement following the administration of rituximab, with nine complete responses and one partial response at 6 months. With a median follow-up of 33.5 months (range 26-45 months), three patients have thus far relapsed. Retreatment with the monoclonal antibody at the same dose and schedule resulted in a new sustained response in all these patients. Rituximab therapy resulted in prolonged B-cell depletion. The ANCA titres decreased significantly in all patients, with eight out of 10 becoming ANCA-negative and three remaining ANCA-negative even after B-cell recovery. Infusion-related side effects were observed in one patient, but were of mild intensity and did not require discontinuation of treatment. CONCLUSIONS: Rituximab is an effective and well-tolerated treatment for patients with ANCA-associated vasculitis and should be strongly considered in severely affected patients who do not respond to standard therapy or in those in whom cytotoxic therapy bears a high risk of morbidity.

The evaluation of a humidifying device for vitreoretinal surgery.

AIM: To study the feasibility of humidifying air during vitreoretinal surgery and measure the water content of air before and after intraocular transit. METHODS: The absolute water content of air was measured in a series of six eyes undergoing fluid-air exchange during macular hole surgery. Infrared absorption spectroscopy was used to determine the water content of the air infusing and exiting each eye. After baseline measurements for each eye were recorded, a second fluid-air exchange was performed and the effect of humidifying the air infusion was documented. The humidifying device used in this study was a prototype adapted from a commercially available respiratory humidifier and enables humidified air to be delivered at a controlled temperature. RESULTS: The water content of air increased following intraocular transit, implying dehydration occurs from the intraocular surfaces. For a standard airline infusion the mean increase in water content of air egressing from an eye was 13.4 mg/l. Humidifying the air reduced the rate of water loss by nearly 90%. CONCLUSIONS: Significant water losses can occur from eyes undergoing fluid-air exchange. Humidifying the infused air can substantially reduce the dehydrating effect during an air exchange. This outcome may have a beneficial effect in reducing cataract formation and visual field defects associated with macular hole surgery.

Increase in the ratio of mitochondrial Bax/Bcl-XL induces Bax activation in human leukemic K562 cell line.

The p53- and Bcl-2-negative leukemic K562 cell line showed resistant to DNA damage-induced Bax activation and apoptosis. The constitutive balanced ratio of Bax/Bcl-XL in K562 mitochondria allowed the formation of active Bax and cytochrome c release from mitochondria in the presence of a BH3-only protein, tBid, in a cell-free system. Bax transfection led to Bax undergoing a conformational change, translocation to mitochondria and homo-oligomerization but not apoptosis in the K562 cell line. After treatment with UV light, while Bcl-XL but not Bax translocated to mitochondria in K562, both Bax and Bcl-XL translocated to mitochondria in the Bax stable transfectant K/Bax cells. The increased ratio of Bax/Bcl-XL in K/Bax mitochondria led to an increased conformationally changed Bax, formation of the homo-multimer of Bax-Bax, and a reduced hetero-dimerization of Bax-Bcl-XL. Increased proportion of active Bax was accompanied with increased percentage of apoptosis. We therefore demonstrate that direct increase in the ratio of mitochondrial Bax/Bcl-XL can induce Bax activation in the p53- and Bcl-2-negative leukemic cells. Increased Bcl-XL translocation and failure in Bax translocation from cytosol to mitochondria play important roles in preventing Bax activation.

A phase II protection study of BB-10010 in patients with high grade non-Hodgkin's lymphoma undergoing intensive chemotherapy.

The aim of this study was to determine whether administration of BB-10010, a synthetic stem cell inhibitor, would allow more intensive chemotherapy to be administered to patients with newly diagnosed high grade NHL. Thirteen patients were randomised to receive BB-10010 concurrently with dose-intensified BEMOP/CA chemotherapy (7 patients) or chemotherapy alone (6 patients). Although the mean neutrophil count of BB-10010 treated patients was higher following cycles 1, 2 and 3 of chemotherapy compared with those receiving chemotherapy alone, there was no difference in the mean number of cycles tolerated, blood component usage and hospital admissions due to infections. No specific toxicity of BB-10010 was identified. Whilst BB-10010 can be administered safely, it does not improve the ability of patients to tolerate intensive chemotherapy for high grade NHL.

Potential mechanisms of leukemia cell resistance to TRAIL-induced apopotosis.

There are many factors contributing to the resistance to TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis. However, it is not clear whether the mechanism of resistance to TRAIL is constitutive or inductive. Therefore, the purpose of this study was to investigate the resistant mechanisms to TRAIL at different levels in the apoptotic pathway. The human T-lymphoblastic leukemic CEM cell line showed more resistant to TRAIL-induced apoptosis compared with the human chronic myeloid leukemic K562 cell line. Lower level of constitutive caspase-8 expression in the CEM cell line led to a poor response to both TRAIL-induced activation of caspase-3 and reduction in the mitochondrial membrane potential (DeltaPsim). There was no significant difference in the constitutive levels of NF-kappaB in CEM and K562 cell lines. However, CEM cells showed a faster response to TRAIL-induced NF-kappaB activation than K562 cells. TRAIL-induced regulation of Bcl-2 family of proteins included an up-regulation in Bcl-2/Bcl-XL and a down-regulation in Bax. IAPs, such as XIAP, cIAP-1, cIAP-2 and Survivin were all up-regulated during the treatment with TRAIL. In summary, our data suggest that the leukemic cells resistance to TRAIL-induced apoptosis might be due to the deficiency in the constitutive caspase-8 expression. Development of potential resistance to apoptosis by TRAIL can occur in both TRAIL-resistant and TRAIL-sensitive leukemic cells.

Humidity devices in vitreoretinal surgery.

PURPOSE: To assess a humidity device used in vitreoretinal surgery. METHODS: Infrared absorption spectroscopy analysis of absolute water content (absolute humidity) was determined for a typical vitreoretinal air infusion line with and without the humidifying device. A variety of experimental laboratory conditions were utilized and designed to mimic those found in the operating room. The effect on physical parameters, such as flow rates and resistance to flow, were also determined. RESULTS: While large bore infusion (LBI) lines had a negligible effect on flow rate and resistance to flow (reduction in infusion pressure), the standard 20 G infusion line (SI) reduced flow rate and infusion pressure by approximately 25%. When used together, the humidifying device and SI reduced flow rate and infusion pressure by one third. The humidifying device was found to add 6.5 mg/L water content to the air infusion line system. Typical operating room (OR) air contains 9.5 mg/L water content, and 43.876 mg/L water content is required to saturate air at body temperature. CONCLUSION: To eliminate dehydration as a cause of intraocular morbidity, one must first saturate the infused air. If the visual field defects and other complications are eliminated, this would be good evidence for saturating infused air. As infusion pressure (flow rate) influences dehydration rate, unless saturated air is used, infusion pressure cannot be considered an independent variable in the analysis of field defects.

Major histocompatibility complex susceptibility genes and immune thrombocytopenic purpura in Caucasian adults.

Immune thrombocytopenic purpura (ITP) is a heterogeneous disorder with wide variability in response rates to treatments including corticosteroids, splenectomy and intravenous immune globulins. The nature of the underlying predisposing causes for this autoimmune disorder are not known. We have HLA typed 71 adult Caucasian patients with chronic primary ITP, and compared the data with 750 control samples. In this association study, we were not able to identify a significant immunogenetic susceptibility factor for ITP with HLA class I and class II alleles. However, it appeared that there might be an association between HLA-A2 and ITP, particularly in female patients, who are the predominantly affected group; and HLA-A2 was also present at increased frequency in patients with chronic ITP progressing to splenectomy. These findings are reviewed in the context of other similar reported HLA studies in ITP. Further studies based on larger groups of patients will be necessary to identify genetic susceptibility factors for this disease.

Liposomal daunorubicin (DaunoXome) in combination with cyclophosphamide, vincristine and prednisolone (COP-X) as salvage therapy in poor-prognosis non-Hodgkins lymphoma.

We treated 33 patients with a variant of the standard 3 weekly CHOP regime, replacing doxorubicin with liposomal daunorubicin (DaunoXome, NeXstar Pharmaceuticals) 120 mg/m2 (COP-X). Eighteen subjects had relapsed/refractory aggressive NHL and 15 had indolent NHL/CLL. Median number of courses received was 4 (1-8). Thirty-two patients were evaluable for efficacy and 26 (81%) responded. 88% of patients with aggressive NHL responded; three (18%) patients achieved complete remission (CR), 12 (70%) achieved partial remission (PR), 1 (6%) patient had stable disease (SD) and 1 (6%) patient progressed through treatment. Median duration of response for patients with aggressive NHL was 3 months. The response rate in indolent NHL/CLL was 73%. Four (27%) patients achieved CR, 7 (46%) PR and 4 (27%) SD. At two years post treatment, 55% of the patients with indolent NHL/CLL remain progression-free, although 4 patients have proceeded to consolidation therapy. Twenty-seven out of 28 (96%) patients developed neutropenia of short duration following one or more of their treatments. Twenty-three patients developed an infection at some stage during therapy (all associated with neutropenia) and required hospitalisation. There were two toxic deaths (infection) both of which occurred in patients who were neutropenic before starting COP-X. Platelet toxicity was mild in patients with normal platelet counts at the commencement of therapy. Alopecia and mucositis were mild. No clinical evidence of myocardial failure was observed. We conclude that the substitution of DaunoXome for doxorubicin in the CHOP regimen to form COP-X provides excellent efficacy against non-Hodgkin's lymphoma. Response durations were short but comparable to those reported with other regimens. COP-X was well tolerated with some suggestion of reduced non-haematological toxicity. The regimen should be considered as an alternative to CHOP with potentially less non-haematological toxicity, particularly cardiac; further studies are required to evaluate the regimen in this context.

Stem cells: progress in research and edging towards the clinical setting.

Mouse embryonic stem cells have been shown to differentiate into a variety of tissues in vitro and in transplantation experiments can produce many different cell types. Multipotent stem cells in adult humans have also shown a high degree of plasticity: haemopoietic stem cells, for example, have been shown to contribute to several other tissues, such as liver. From these simple observations there has been considerable extrapolation into the use of such putative totipotent stem cells in the clinical setting, with the development of 'designer' tissue engineering, whose aim is to create large tissues or even whole organs for clinical use. In practical terms, however, there are many limitations and difficulties and clinical use has been restricted to a very few settings, eg the use of fetal cells in Parkinson's disease. Nonetheless, there is enormous potential in this area, and also in the application of embryonic or adult stem cells as carriers for gene therapy; but the limitations of such treatment, in particular the stability of manipulated cells, and the problems of ageing and Ooncogenicity, not to mention a host of ethical and regulatory issues, all need to be considered.

c-IAP1 blocks TNFalpha-mediated cytotoxicity upstream of caspase-dependent and -independent mitochondrial events in human leukemic cells.

Tumor necrosis factor-alpha (TNFalpha) mediates cytochrome c release from mitochondria, loss of mitochondrial membrane potential (DeltaPsim) and apoptosis in sensitive leukemic cells. In the present study, by using the human leukemic U937 cell line, we demonstrate that the cytochrome c release is caspase-8-dependent and can be blocked by an inhibitor of caspase-8, Z-Ile-Glu (OMe)-Thr-Asp(OMe)-fluoromethyl ketone (Z-IETD.fmk), or a pan caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD.fmk). However, TNFalpha-mediated loss of DeltaPsim was not inhibited by caspase inhibitors. The apoptotic process was blocked by either Z-IETD.fmk or Z-VAD.fmk in cells with lower DeltaPsim. U937 cells with stable transfection of the cellular inhibitor of apoptosis protein 1 (c-IAP1) are resistant to TNFalpha-induced activation of caspases, Bid cleavage, cytochrome c release and DeltaPsim collapse. In addition, both c-IAP1 and XIAP were not up-regulated upon prolonged exposure to TNFalpha. In contrast, there was a caspase-dependent cleavage of XIAP, but not c-IAP1, during treatment with TNFalpha for 7 days. These results demonstrate that c-IAP1 blocks TNFalpha signaling at a level controlling both activation of caspase-8 and a signal to cause loss of DeltaPsim. The sensitive U937 cell line failed to acquire resistance and gain a self-protecting advantage against apoptosis, upon induction of c-IAP1 expression.

Bax translocation is crucial for the sensitivity of leukaemic cells to etoposide-induced apoptosis.

Bax translocation from cytosol to mitochondria is believed to be a crucial step for triggering cytochrome c release from mitochondria. However, it is unclear whether Bax translocation is associated with Bax induction by DNA damaging agents. The induction of Bax in response to DNA damaging agents has been considered to be linked with p53. In this study, we used the p53 negative human chronic myeloid leukaemia K562 cell line. Bax up-regulation occurred at the whole cell level after DNA damage induced by etoposide. However, after incubation with etoposide, Bax failed to translocate to mitochondria and as a result, the apoptotic process was blocked. A Bax stable transfectant, the K/Bax cell line, expressed more Bax protein in the cytosol, mitochondria and nuclei. This Bax overexpression induced cytochrome c release, a reduction of cytochrome c oxidase activity and mitochondrial membrane potential (Delta(Psi)m). However, Bax-induced apoptosis was blocked downstream of mitochondria in K562 cells. The increased levels of mitochondrial Bax sensitized cells to etoposide-induced activation of caspases-2, -3 and -9 and apoptosis. However, after transient transfection with the Apaf-1 gene, K/Bax cells were sensitized to etoposide-induced caspase activation and apoptosis to a larger extent compared with Bax or Apaf-1 transfection alone. We therefore conclude that two mechanisms contribute to the resistance of K562 cells to etoposide-induced apoptosis; firstly failure of Bax targeting to mitochondria and, secondly, deficiency of Apaf-1. Uncoupling of Bax translocation from Bax induction can occur in response to etoposide-induced DNA damage.

Apaf-1 protein deficiency confers resistance to cytochrome c-dependent apoptosis in human leukemic cells.

The human leukemia cell lines K562, CEM, CEM/VLB(100), human leukemic blasts, and the bladder cancer J82 cell line have different sensitivities to UV light-induced apoptosis. It is reported that resistance to UV light-induced apoptosis occurs at a point in the apoptotic pathway upstream of caspase-3 but downstream of mitochondrial cytochrome c release. It is demonstrated that the block is due to deficiency of Apaf-1, a critical member of the apoptosome. Sensitivity to apoptosis was independent of caspase-9b or XIAP (inhibitors of apoptosis proteins) expression or levels of procaspase-9. Transfection of Apaf-1 conferred sensitivity to apoptosis in resistant cells. Apaf-1 deficiency may constitute a significant mode of resistance to apoptosis in human leukemia.

Quantitative determination of apoptosis on leukemia cells by infrared spectroscopy.

Quantitation of apoptotic cell death in vivo has become an important issue for patients with acute leukemia. We describe herein a new analytical method, based on infrared (IR) spectroscopy, to estimate the percentage of apoptotic leukemic cells in two different cell lines (CEM and K562), induced with etoposide (VP-16). As the percentage of apoptosis increases, the protein structure shifts from dominantly beta-sheet to unordered (random coil), the overall lipid content increases and the amount of detectable DNA decreases. These changes can be directly related to the percentage of apoptosis as determined by two standard reference methods: flow cytometry and DNA ladder formation. The correlation between the significant IR spectral changes and the percentage of apoptotic leukemia cells in the two cell lines was optimal up to 24 h following etoposide treatment (r = 0.99 for CEM cells and r = 0.96 for K562 cells). Furthermore, IR spectroscopy is able to detect apoptotic changes in these cells already after 4 h treatment with VP-16, compared to flow cytometry which needs 6 h to observe significant changes. Our study suggests that IR spectroscopy may have potential clinical utility for the early, fast and reagent free assessment of chemotherapeutic efficacy in patients with leukemia.

Trail-induced apoptosis in Type I leukemic cells is not enhanced by overexpression of bax.

We have previously shown that Bax translocation was crucial in TNFalpha or etoposide-induced apoptosis. Overexpression of Bax sensitized chronic myeloid leukemic K562 cells to etoposide-induced apoptosis. Treatment with TNF-related apoptosis-inducing ligand (TRAIL) induces a loss of mitochondrial membrane potential (DeltaPsim), cytochrome c release from mitochondria, activation of caspases-8, -9, and -3, and cleavage of Bid in the K562 cell line. Bax failed to sensitize K562 cells to TRAIL-induced apoptosis. TRAIL did not induce Bax expression and/or translocation from cytosol to mitochondria in the K562 cell line. However, 100 microM Z-VAD.fmk, a pan caspase inhibitor, completely blocked TRAIL-initiated mitochondrial alterations and cleavages of caspases and Bid. We propose that TRAIL-induced apoptosis in K562 cells is via Type I apoptotic signal pathway. Bax translocation is not essential for TRAIL-induced cytochrome c release and DeltaPsim collapse in the Type I cells.